Infection or inflammation of the male reproductive tract often lead to permanent infertility. Yet, one of the most understudied aspects of male reproductive physiology is the ability of the excurrent duct to protect maturing spermatozoa against an autoimmune response, while initiating very efficient immune activation against pathogens. The present application is based on our new discovery that a dense dendritic cell network populates the epididymis. We showed that epididymal dendritic cells (eDCs) establish intimate interactions with the epididymal mucosa and can send dendritic projections between epithelial cells toward the lumen. eDCs have the ability to present antigens to CD4+ and CD8+ T cels in vitro. We hypothesize that eDCs can interact with the luminal content, including spermatozoa, and regulate the balance between immune tolerance and inflammation. Aim 1a wil characterize eDCs using high resolution imaging in CD11c-EYFP and CX3CR1-GFP transgenic mice, comparative flow cytometry analysis and antigen-presenting assays before, during and after puberty. We postulate that the eDC network is reorganized during puberty, when sperm begin populating the excurrent duct. Aim 1b wil examine the regional specificities of eDC subsets in the context of the dual function of the epididymis. eDCs will be isolated from the proximal epididymal region (primarily involved in sperm maturation and protection against an autoimmune response), and distal region (more susceptible to infections by ascending pathogens). Comparative flow cytometry analysis and antigen-presenting capability will be determined in proximal and distal eDC populations. Aim 2 wil determine the role of eDCs in the homeostatic and inflamed mouse epididymis, using conventional fluorescence and intravital multiphoton microscopy, and flow cytometry analysis. Aim 2a wil test the hypothesis that eDCs have the ability to sample and process luminal antigens including those present in sperm. Aim 2b will analyze the behavior of eDCs in the epididymis after injection with LPS (to mimic infections), vasectomy (downstream blockade of luminal flow) or efferent duct ligation (upstream blockade of luminal flow). The goal of this application is to characterize the nature and functions of the eDC network to provide new frameworks for the treatment of male immunological infertility (which is a major public health issue) and, ultimately, for the control of male fertility. PUBLIC HEALTH RELEVANCE: Spermatozoa acquire their fertilizing capacity during their transit through the epididymis, a long tube located downstream of the testis. Our laboratory studies how dendritic cells, which are among the most potent regulators of the immune system, work together with other cells lining the epididymal tube to protect spermatozoa against pathogens and autoimmunity. These results will provide new frameworks for the treatment of immunological male infertility.